Further evidence has been obtained for cooperative interactions between head and tail domains of Acanthamoeba myosin II that may be related to the "long-range" regulation of ATPase and motility activities of the N- terminal head domain by phosphorylation at the C-terminal tip of the tail domain. Specifically, differential scanning calorimetry reveals highly cooperative interactions between the head and tail domain that are uncoupled by binding of MgATP to the head domain. Also, MgATP bound to the head domain accelerates the rate of papain cleavage at a site 9-Kda from the C-terminus of filaments of phosphorylated myosin II. Myosin I heavy chain kinase (which activates myosin I by phosphorylation of a single residue in the N-terminal domain of the heavy chain) is itself activated by autophosphorylation which is stimulated by acidic phospholipids and plasma membranes. Further studies of the activation of myosin I heavy chain kinase by plasma membranes has shown that activation occurs rapidly upon binding to membranes and precedes autophosphorylation. Thus, there appear to be two mechanisms for stimulating kinase activity in vitro. Inhibitory antibodies specific for myosin IC have been found to inhibit the function of contractile vacuoles in Acanthamoeba castellanii exposed to hypotonic media. We had previously shown that myosin IC is the only myosin I isoform associated with the contractile vacuole membrane. This is the first evidence for a specific function for a membrane-associated myosin I.